PROJECT SUMMARY/ABSTRACT Liver cancers with increasing frequency in the world have become the second cause of cancer related death; however an important gap in current knowledge remains the molecular fingerprint and organization of hepatocarcinoma/hepatoma cells which might allow their specific targeting. We found that the expression level of two proteins, VDAC2 and Bak, that are central to a mitochondrial apoptosis pathway, is low in normal hepatocytes and is increased during hepatic tumor progression. Furthermore, we also found that the pro- apoptotic BH3-only Bcl-2 family protein, Bid induces mitochondrial apoptosis more efficiently in hepatocarcinoma than in normal liver. This difference can be eliminated by VDAC2 silencing in hepatocarcinoma cells, and by VDAC2 expression in normal hepatocytes but only if Bak isn?t knocked out. The potential medical significance of the VDAC2-Bak heterogeneity is supported by human databases that show upregulation of VDAC2 and Bak proteins in liver cancer. We have also documented both subcellular and cell- to-cell differences in the tBid-Bak pathway. Finally, we have shown at least in cell culture, that hepatocarcinoma cells can be selectively killed through the tBid-Bak pathway (using its activators and suppressors of its inhibitor, Mcl-1 while normal hepatocytes are spared. We here, postulate that the heterogeneity in VDAC2 and/or Bak abundance in the liver are important for hepatoma/ hepatocarcinoma (1) growth and (2) targeting by the combination of an Mcl-1 inhibitor drug and a cell permeable hydrocarbon stapled Bid BH3 peptide. Heterogeneity is considered (1) within the cells among individual mitochondria, (2) in each cell type among single cells, and (3) among the different cell types. To test our hypothesis we have established a combination of genetic targeting, tumorigenesis in mouse, and biochemical and microscopic imaging approaches. In the study we will focus on (1) assessing heterogeneity of VDAC2, Bak and Bak-mediated OMM permeabilization in hepatocarcinoma cells and normal hepatocytes and their dependence on VDAC2 expression and mitochondrial fusion; (2) determining whether increased expression of VDAC2 through upregulation of Bak affects hepatoma/hepatocarcinoma progression; (3) testing if activation of Bak by treatment with Mcl-1 inhibitor (S-63845) and a cell permeable hydrocarbon stapled Bid peptide can be used to kill hepatocyte-derived tumors without damaging normal hepatocytes; and (4) determining additional VDAC2-dependent proteins in hepatic tumor cells and to evaluate their heterogeneity in the liver and their relevance for hepatoma/hepatocarcinoma growth. By addressing these points, our study will provide clues to the contribution of mitochondrial heterogeneity to hepatic tumorigenesis and test a novel tumor-selective targeting approach.